The present invention relates to the isolation, purification and characterization of proteins mediating switch recombination. The present invention further relates to the microbial production via recombinant DNA technology of recombination protein SRTA-70, a member of the proteins mediating switch recombination. The present invention further relates to the use of these proteins as therapeutically active agents in immune response modulation, specifically, in augmentation and suppression of the immune response.
Higher eukaryotes produce immunoglobulins (Ig) of diffent classes, which are defined by the constant region (C) of the heavy (H) chain. Upon stimulation by antigen expression of the early IgM class changes to that of another H chain class. This switch from one H chain class to another, named simply xe2x80x9cclass switchingxe2x80x9d, occurs via DNA recombination. Switch recombination imprecisely joins two so-called switch (S) regions, which lie upstream of the H chain genes and contain highly repetitive sequences (for reviews see Esser and Radbruch, Annu. Rev. Immunol. 8, 717-735 [1990] and Harriman et al., Annu. Rev. Immunol. 11, 361-384 [1993]). The recombination mechanism for most class switching events can be described by the loop-excision model (Jxc3xa4ck et al., Proc. Natl. Acad. Sci. USA 85, 1581-1585 [1988]). The biochemistry of the class switch recombination process, however, remains largely unknown.
In order to study the mechanism of class switch recombination an assay that measures DNA-transfer activity was devised which makes use of two S (Sxcexc and Sxcex32b) regions, cloned into two different, largely non-homologous vectors (FIG. 1). Using this assay three proteins in the S-Region Transfer Activity (SRTA) were identified: B23 (nucleophosmin), poly (ADP) ribose polymerase (PARP) and a novel 70-KDa protein SRTA-70.
Thus, in a first aspect of this invention, there are provided SRTA-70 proteins, specifically recombinantly produced SRTA-70 protein. The term xe2x80x9crecombinantly produced SRTA-70 proteinxe2x80x9d refers to the protein of SEQ ID No. 1 or any protein or polypeptide having an amino acid sequence which is substantially homologous to the amino acid sequence SEQ ID No. 1 and further having the biological activities of the protein of SEQ ID No. 1.
As used hereinbefore the term xe2x80x9csubstantially homologousxe2x80x9d means that a particular subject sequence, for example, a mutant sequence, varies from a reference sequence by one or more substitutions, deletions, or additions, the net effect of which does not result in an adverse functional dissimilarity between the reference and subject sequences. For purposes of the present invention, sequences having greater than 95 percent homology, equivalent biological activity and equivalent expression characteristics are considered substantially homologous. For purposes of determining homology, truncation of the sequence should be disregarded. Sequences having lesser degrees of homology, comparable bioactivity, and equivalent expression characteristics, e.g., fragments of the amino acid sequence SEQ ID No: 1 are considered substantial equivalents.
As used herein the term recombinantly produced SRTA-70 protein includes proteins modified deliberately, as for example, by addition of specific sequences that preferably bind to an affinity carrier material. Examples of such sequences are sequences containing at least two adjacent histidine residues (see in this respect European Patent No. 282 042). Such sequences bind selectively to nitrilotriacetic acid nickel chelate resins (Hochuli and Dxc3x6beli, Biol. Chem. Hoope-Seyler 368, 748 [1987]; European Patent No. 253 303). SRTA-70 proteins which contain such a specific sequence can, therefore, be separated selectively from the remaining polypeptides. The specific sequence can be linked either to the C-terminus or the N-terminus of the SRTA-70 protein.
There are further provided isolated DNA sequences encoding SRTA-70 proteins or fragments thereof. Specifically, the DNA sequences of this invention are defined to include the nucleotide sequence SEQ ID No: 2 or a fragment thereof or any DNA sequence which is substantially homologous to the nucleotide sequence SEQ ID No: 2 or a fragment thereof.
As used hereinbefore the term xe2x80x9csubstantially homologousxe2x80x9d, means that a particular subject sequence, for example, a mutant sequence, varies from a reference sequence by one or more substitutions, deletions, or additions, the net effect of which does not result in an adverse functional dissimilarity between the reference and subject sequences. For purposes of the present invention, DNA sequences having greater than 95 percent homology, encoding equivalent biological properties, and showing equivalent expression characteristics are considered substantially homologous. For purposes of determining homology, truncation of the DNA sequence should be disregarded. Sequences having lesser degrees of homology, encoding comparable bioactivity, and showing equivalent expression characteristics, e.g., fragments of the nucleotide sequence SEQ ID No: 2 are considered substantial equivalents. Generally, homologous DNA sequences can be identified by cross-hybridization under standard hybridization conditions of moderate stringency.
There are also provided vectors and expression vectors containing the DNA sequences of the present invention, hosts containing such vectors for the production of SRTA-70 proteins, and processes for the production of such DNA sequences, recombinant vectors and host cells.
Methods for the expression, isolation and purification of the SRTA-70 proteins are also provided.
The following steps outline the methods for recombinantly expressing the SRTA-70 proteins.
1) Cloning of DNA Sequences Encoding SRTA-70 Proteins
DNA sequences encoding SRTA-70 proteins can be cloned using a variety of techniques. Using the methods described in this application cDNAs encoding SRTA-70 proteins or fragments thereof can be produced. These cDNAs can be isolated and amplified by PCR technique using oligodeoxynucleotide DNA primers by conventional techniques.
The cDNA (SEQ ID No: 2) encoding the amino acid sequence SEQ ID No:1 is obtained using the DNA primers described in the examples. By using conventional technique, this cDNA has been isolated from a mouse spleen cDNA library.
The cDNA may be obtained not only from cDNA libraries, but by other conventional techniques, e.g., by cloning genomic DNA, or fragments thereof, purified from the desired cells. These procedures are described by Sambrook et al., in xe2x80x9cDNA Cloning: A Practical Approachxe2x80x9d, Vol. I and II, D. N. Glover, ed., 1985, MRL Press, Ltd., Oxford, U. K.; Benton and Davis, Science 196, 180-182 [1977]; Grunstein and Hogness, Proc. Nat. Acad. Sci. 72, 3961-3965 [1975]; and Maniatis et al., in xe2x80x9cMolecular Cloning-A Laboratory Manualxe2x80x9d, Cold Spring Harbor Laboratory [1989].
To obtain the cDNA encoding the SRTA-70 proteins cDNA libraries are screened by conventional DNA hybridization techniques by the methods of Benton and Davis, supra, or Grunstein and Hogness, supra, using radioactive SRTA-70 gene fragments. Clones which hybridize to the radioactive gene fragments are analyzed, e.g., by restriction endonuclease cleavage or agarose gel electrophoresis. After isolating several positive clones the positive insert of one clone is subcloned, e.g., into phagemids, and sequenced by conventional techniques.
Clones derived from genomic DNA may contain regulatory and intron DNA regions in addition to coding regions; clones derived from cDNA will not contain intron sequences. In the molecular cloning of the gene from genomic DNA, DNA fragments are generated, some of which will encode the desired gene. The DNA may be cleaved at specific sites using various restriction enzymes. Alternatively, one may use DNAse in the presence of manganese to fragment the DNA, or the DNA can be physically sheared, as for example, by sonication. The linear DNA fragments can then be separated according to size by standard techniques, including but not limited to, agarose and polyacrylamide gel electrophoresis and column chromatography.
Whatever the source, the DNA sequence encoding SRTA-70 proteins may be molecularily cloned into a suitable vector for propagation of the DNA by methods known in the art. Any commercially available vector may be used. For example, the DNA may be inserted into a pBluescript SKxe2x88x92 vector. Appropriate vectors for use with bacterial hosts are described by Pouwels et al., in xe2x80x9cCloning Vectors: A Laboratory, Manualxe2x80x9d, 1985, Elsevier, N.Y. As a representative but nonlimiting example, useful cloning vectors for bacterial use can comprise a selectable marker and a bacterial origin of replication derived from commercially available plasmids which are in turn derived from the well known cloning vector pBR322 (ATCC 37017). Such commercial vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and pGEM1 (Promega Biotec, Madison, Wis., USA).
The DNA sequences encoding SRTA-70 proteins inserted in these commercially available vectors can be verified by methods known in the art, e.g., by standard nucleotide sequencing techniques.
DNA sequences that code for SRTA-70 proteins from mammals other than mice may be used herein. Accordingly, while specific DNA has been cloned and sequenced in relation to the DNA sequence in mouse cells, any mammalian or vertebrate cell potentially can be used as the nucleic acid source of the SRTA-70 protein.
2) Production of SRTA-70 Proteins
Cloned DNA sequences that code for SRTA-70 proteins can be expressed in hosts to enable the production of these proteins with greater efficiency. Techniques for these genetic manipulations are specific for the different available hosts and are known in the art.
For expression of SRTA-70 proteins in hosts, in principle, all vectors which replicate and express DNA sequences encoding the SRTA-70 proteins in the chosen host are suitable. Expression vectors suitable for use in prokaryotic host cells are mentioned, for example, in the textbooks xe2x80x9cMolecular Cloningxe2x80x94A Laboratory Manualxe2x80x9d, Cold Spring Harbor Laboratory [1982] and [1989], of Maniatis et al. Examples of other vectors are plasmids of the pDS family (Bujard et al., Methods in Enzymology, eds. Wu and Grossmann, Academic Press, Inc., Vol. 155, 416-433 [1987]).
Such prokaryotic expression vectors which contain the DNA sequences coding for the SRTA-70 proteins operatively linked with an expression control sequence can be incorporated using conventional methods into any suitable prokaryotic host cell. The selection of a suitable prokaryotic host cell is determined by different factors which are well-known in the art. Thus, for example, compatibility with the chosen vector, toxicity of the expression product, expression characteristics, necessary biological safety precautions and costs play a role and a compromise between all of these factors must be found.
Suitable prokaryotic host organisms include gram-negative and gram-positive bacteria, for example E. coli and B. subtilis strains. Examples of prokaryotic host organisms are E. coli strain M15 (described as strain OZ 291 by Villarejo et al. in J. Bacteriol. 120, 466-474 [1974] and E. coli W3110 [ATCC No. 27325]). In addition to the aforementioned E. coli strains, however, other generally accessible E. coli strains such as E. coli 294 (ATCC No. 31446) and E. coli RR1 (ATCC No. 31343) can also be used. In a preferred embodiment of the present invention E. coli M15 is used as the host organism.
Expression vectors suitable for use in yeast cells are described in xe2x80x9cGuide to yeast genetics and molecular biologyxe2x80x9d, Guthrie and Fink, eds., Methods in Enzymology, Academic Press, Inc., Vol. 194 (1991) and xe2x80x9cGene expression technologyxe2x80x9d, Goeddel, ed., Methods in Enzymology, Academic Press, Inc., Vol. 185 [1991]. Examples of suitable yeast cells are Saccharomyces cerevisiae, Pichia pastoris, Hansenula polymorpha, Schizosaccharomyces pombe cells. An overview on various yeast expression systems is given by Romanos et al., Yeast, Vol. 8, 423-488 [1992].
The transformation with the yeast expression vectors is carried out as described by Klebe et al., Gene, Vol. 25, 333-341 [1983].
Plants can also be used as hosts for the production of SRTA-70 protein of the present invention. Transfer of the DNA sequence coding for the SRTA-70 protein may be achieved by a variety of methods (for review see Potrykus and Spangenberg, eds., Gene transfer to plants. A laboratory manual, Springer Verlag, Heidelberg, Germany [1995]), whereby the DNA sequence for the SRTA-70 protein is integrated into the chromosome of the host plants. Over-expression of the SRTA-70 protein may be achieved, for example, by transforming a plant host with the DNA sequence coding for the SRTA-70 protein. Examples of plant hosts for the production of SRTA-70 protein include, but are not limited to maize (Zea mays, Ishida et al., Nature Biotechnology 14, 745-750 [1996]), flax (Linum usitatissimum, Dong and Mchughen, Plant Sci. 88 (1), 61-71 [1993]), soybean (Glycine max, Christou et al., Tibtech 8, 145-151 [1990]), alfalfa or tobacco.
The manner in which the expression of the SRTA-70 proteins is carried out depends on the chosen expression vector host cell system.
Usually, the prokaryotic host cells which contain a desired expression vector are grown under conditions which are optimal for the growth of the prokaryotic host cells. At the end of the exponential growth, when the increase in cell number per unit time decreases, the expression of the desired SRTA-70 protein is induced, i.e., the DNA coding for the desired SRTA-70 protein is transcribed and the transcribed mRNA is translated. The induction can be carried out by adding an inducer or a derepressor to the growth medium or by altering a physical parameter, e.g., a change in temperature. For example, the expression can be controlled by the lac repressor.
By adding isopropyl-xcex2-D-thiogalactopyranoside (IPTG), the expression control sequence is derepressed and the synthesis of the desired protein is thereby induced.
The yeast host cells which contain a desired expression vector are grown under conditions which are optimal for the growth of the yeast host cells. A typical expression vector contains the promoter element, which mediates the transcription of mRNA, the protein coding sequence, a ribosomal binding site for effective translation. Additional elements may include terminator, signal, and upstream activating sequences.
The yeast cells are grown as described by Sherman in xe2x80x9cGuide to yeast genetics and molecular biologyxe2x80x9d, Guthrie and Fink, eds., Methods in Enzymology, Academic Press, Inc., Vol. 194, 3-21 [1991].
The baculovirus-insect cell vector system can also be used for the production of the SRTA-70 proteins of the present invention (for review see Luclow and Summers, Bio Technology 6, 47-55 [1988]). The SRTA-70 proteins produced in insect cells infected with recombinant baculovirus can undergo post-translational processing including but not limited to N-glycosylation (Smith et al., Proc. Nat. Scad. Sci. USA 82, 8404-8408) and O-glycosylation (Thomsen et al., 12. International Herpesvirus Workshop, University of Philadelphia, Pa.).
Mammalian cells can also be used as hosts for the recombinant production of SRTA-70 proteins. Suitable mammalian host cells include but are not limited to human Hela, H9 and Jurkat cells, mouse NIH3T3 and C127 cells, CV1 African green monkey kidney cells, quail QC1-3 cells, Chinese hamster ovary (CHO) cells, mouse L cells and the COS cell lines.
Expression vectors suitable for use in mammalian host cells include but are not limited to pBC12MI, pBC12BI, pSV2dhFr, p91023(B), pcDNA3, pcDV1, pRSVcat, pGA291, pGA293, pGA296, pBC12/HIV/IL-2 and PGA300. Such vectors are preferably introduced into suitable mammalian host cells by transfection.
Usually, the mammalian host cells which contain a desired expression vector are grown under conditions which are optimal for the growth of the mammalian host cells. A typical expression vector contains the promoter element, which mediates the transcription of mRNA, the protein coding sequence, and the signals required for efficient termination and polyadenylation of the transcript. Additional elements may include enhancers and intervening sequences bounded by spliced donor and acceptor sites.
Most of the vectors used for the transient expression of a given coding sequence carry the SV40 origin of replication, which allows them to replicate to high copy numbers in cells (e.g. COS cells) that constitutively express the T antigen required to initiate viral DNA synthesis. Transient expression is not limited to COS cells. Any mammalian cell line that can be transfected can be utilized for this purpose. Elements that control a high efficient transcription include the early or the late promoters from SV40 and the long terminal repeats (LTRs) from retroviruses, e.g. RSV, HIV, HTLVI. However, also cellular signals can be used (e.g. human xcex2-actin-promoter).
Alternatively stable cell lines carrying a gene of interest integrated into the chromosome can be selected upon co-transfection with a selectable marker such as gpt, dhfr, neomycin or hygromycin.
Now, the transfected gene can be amplified to express large quantities of a foreign protein. The dihydrofolate reductase (DHFR) is a useful marker to develop lines of cells carrying more than 1000 copies of the gene of interest. The mammalian cells are grown in increasing amounts of methotrexate. Subsequently, when the methotrexate is withdrawn, cell lines contain the amplified gene integrated into the chromosome.
Transgenic animal vector systems can also be used for the production of SRTA-70 proteins of the present invention (for review see Pinkert, Transgenic animal technology: a laboratory handbook, Academic Press, San Diego [1993]). Using specific signal sequences the desired SRTA-70 protein can also be secreted into the milk of the animal (for examples see Drohan et al., J. Cell. Biochemistry 17a, 38-38 [1993]; Lee et al., Appl. Biochem. Biotechnol. 56, 211-222 [1996]) thus allowing the use of the milk as a source for SRTA-70 protein.
For the isolation of small amounts of SRTA-70 proteins expressed in prokaryotic host cells for analytical purposes, e.g., for polyacrylamide gel electrophoresis, the host cells can be disrupted by treatment with a detergent, e.g., sodium dodecyl sulphate (SDS). Larger quantities of SRTA-70 protein can be obtained by mechanical (Charm et al., Meth. Enzymol. 22, 476-556 [1971]), enzymatic (lysozyme treatment) or chemical (detergent treatment, urea or guanidinium hydrochloride treatment, etc.) treatments followed by use of known methods, e.g., by centrifugation at different gravities, precipitation with ammonium sulphate, dialysis (at normal pressure or at reduced pressure), preparative isoelectric focusing, preparative gel electrophoresis or by various chromatographic methods such as gel filtration, high performance liquid chromatography (HPLC), ion exchange chromatography, reverse phase chromatography and affinity chromatography (e.g., on Sepharose(copyright) Blue CL-6B).
Preferably, the SRTA-70 proteins expressed in prokaryotic host cells are obtained after Ni-Agarose affinity chromatography followed by gel filtration.
The SRTA-70 proteins expressed in mammalian host cells or in the baculovirus-insect cell vector system can be isolated from the host cell medium using standard protein purification methods.
The SRTA-70 proteins can be used as therapeutically active agents in immune response modulation, specifically, in augmentation and suppression of the immune system.
Furthermore, the SRTA-70 proteins can be used as mediators of protein-protein interactions to retrieve other proteins involved in DNA recombination and repair, especially class switch recombination, and other metabolic processes. SRTA-70 proteins can serve as hooks to pull other relevant proteins out of cell extracts, and allow cloning the respective genes. SRTA-70 proteins can also be used for identification of compounds inhibiting or boosting the function of SRTA-70 proteins and proteins and nucleic acids interacting with SRTA-70 proteins (agonists or antagonists).
Antibodies can also be raised against the SRTA-70 proteins of the present invention. These antibodies can be used in a well-known manner for diagnostic or therapeutic purposes as well as for localisation and purification purposes. Such antibodies can be produced by injecting a mammalian or avian animal with a sufficient amount of a vaccine formulation comprising a SRTA-70 protein of the present invention and a compatible pharmaceutical carrier to elicit the production of antibodies against said receptor. The appropriate amount of the SRTA-70 proteins which would be required would be known to one of skill in the art or could be determined by routine experimentation. SRTA-70 specific antibodies may also be selected from phage, viral, or bacterial antibody libraries. As used in connection with this invention the term xe2x80x9cpharmaceutical carrierxe2x80x9d can mean either the standard compositions which are suitable for human administration or the typical adjuvants employed in animal vaccinations.
Suitable adjuvants for the vaccination of animals include but are not limited to Freund""s complete or incomplete adjuvant (not suitable for human or livestock use). Adjuvant 65 (containing peanut oil, mannide monooleate, aluminum phosphate and alum; surfactants such as hexadecylamine, octadecylamine, lysolecithin, dimethyldioctadecylammonium bromide, N1-N-dioctadecyl-Nxe2x80x2-N-bis(2-hydroxyethylpropanediamine), methoxyhexy-decylglycerol, and pluronic polyols; polyanions such as pyran, dextran sulfate, poly IC, polyacrylic acid, carbopol; peptides such as muramyl dipeptide, dimentylglycine, tuftsin; and oil emulsions. The SRTA-70 proteins could also be administered following incorporation into liposomes or other microcarriers, or after conjugation to polysaccharides, other proteins or other polymers or in combination with Quil-A to form xe2x80x9cIscomsxe2x80x9d (immuno-stimulating complexes) (Morein et al., Nature 308, 457 [1984]).
Typically, the initial vaccination is followed some weeks later by one or more xe2x80x9cboosterxe2x80x9d vaccinations, the net effect of which is the production of high titers of antibodies against the SRTA-70 proteins which can be harvested in the usual way.
Another method consists in using the well-known Koehler and Milstein technique for producing monoclonal antibodies. In order to find out different monoclonal antibodies which are directed against the same antigen but against different epitopes, the method of Stxc3xa4hli et al. (J. of Immunological Methods 21, 297-304 [1980]) can be used.
The antibodies against the SRTA-70 proteins are useful for determination of the expression (over- and underexpression) of SRTA-70 protein. As set forth below altered features of SRTA-70 expression may lead to cancer and allergy.
Various methods which are generally known can be employed in the determination of SRTA-70 protein.
In one such procedure known amounts of a sample to be assayed, radio-labeled SRTA-70 protein and unlabeled SRTA-70 protein are mixed together and allowed to stand. The antibody/antigen complex is separated from the unbound reagents by procedure known in the art, e.g., by treatment with ammonium sulphate, polyethylene glycol, second antibody either in access or bound to an insoluble support, dextran-coated charcoal and the like. The concentration of the labeled SRTA-70 protein is determined in either the bound or unbound phase and the SRTA-70 content of the sample can then be determined by comparing the level of labeled component observed to a standard curve in a manner known per se.
Another suitable method is the xe2x80x9cDouble-Antibody-Sandwich-Assayxe2x80x9d. According to this assay the sample to be tested is treated with two different antibodies. One of these antibodies is labeled and the other is coated on a solid phase.
Suitable solid phases are organic and inorganic polymers [amylases, dextrans, natural or modified celluloses, polyacrylamides, agaroses, magnetite, porous glass powder, polyvinylidene fluoride (Kynar) and latex], the inner wall of test vessels (test tube, titer plates or cuvettes of glass or artificial material) as well as the surface of solid bodies (rods of glass and artificial material, rods with terminal thickening, rods with terminal lobes or lamellae). Spheres of glass and artificial material are especially suitable solid phase carriers.
Suitable labels are enzymes, e.g., peroxidase, radio-labels or fluorescence-labels.
Different antibodies can, e.g., be achieved by immunizing different animals, e.g., sheep and rabbits.
The methods for the determination of SRTA-70 protein as described above can be conducted in suitable test kits comprising in a container antibodies against SRTA-70 protein elicited by a SRTA-70 protein of the present invention.
The isolated DNA sequences encoding SRTA-70 proteins are useful to make probes for assaying the status of the natural SRTA-70 gene (i.e. mutations, deletions, rearrangements, amplifications etc.), or its expression (over- and underexpression). This could be relevant to class switch recombination, DNA recombination and repair in general, and other processes. There might be diseases, that are linked to altered features of SRTA-70, for example, DNA repair deficiencies leading to cancer; class switch aberrations or redirections towards allergy-causing IgE expression, and general B lymphocyte hypo- or hyperactivity.
The isolated DNA sequences encoding SRTA-70 proteins can also be used to generate antisense RNA to alter expression of the endogeneous SRTA-70 gene.
The isolated DNA sequences encoding SRTA-70 proteins, cloned into appropriate vectors, can be used for overexpression of the gene in target cells. This could create cellular models for the effect of altered SRTA-70 expression on class switch recombination, DNA recombination and repair, related processes, and general B cell function. This could allow to search for compounds which (counter-) regulate SRTA-70 protein expression.
Finally, the isolated DNA sequences encoding SRTA-70 proteins can be used to generate knockout mice. Such mice may have altered class switch recombination, and/or altered DNA recombination and repair processes in general. Such mice could be used as models for recombination-related diseases (cancer, allergies etc.), as well as for immune disorders related to B cell function, and as models for the respective therapeutic trials.
It has also been discovered that protein B23 has DNA recombination functions. Thus, the present invention provides in addition the use of protein B23 as a mediator of protein-protein interactions to retrieve other proteins involved in DNA recombination and repair, especially class switch recombination, and other DNA metabolic proceses. Protein B23 could serve as a hook to pull other relevant proteins out of cell extracts. and allow cloning the respective genes. Protein B23 could also be used for screening of compounds inhibiting or boosting the function of B23 (agonists or antagonists) and proteins and nucleic acids interacting with B23.
Isolated DNA sequences encoding protein B23 are useful to make probes for assaying the status of the natural B23 gene (i.e. mutations, deletions, rearrangements, amplifications etc.), or its expression (over- and under-expression). This could be relevant to class switch recombination, DNA recombination and repair in general, and other processes. There might be diseases, that are linked to altered features of B23 (for example DNA repair deficiencies leading to cancer; class switch aberrations or redirections towards allergy-causing IgE expression).
Further it has been discovered that protein PARP interacts with proteins B23 and SRTA-70. Thus, the present invention provides further the use of protein PARP as a regulator of the recombinative (DNA metabolic) functions of B23 and SRTA-70 and thereby involvement of PARP through interaction with SRTA-70 and B23 in class switch recombination; DNA repair and recombination, and related processes.
Having now generally described this invention, the same will become better understood by reference to the specific examples, which are included herein for purpose of illustration only and are not intended to be limiting unless otherwise specified, in connection with the following figures: